A biosensor contains a biological component (e.g., enzyme, antibody, DNA/RNA, aptamer) coupled to a transducer, which is typically a physical sensor, such as a microcantilever, or a chemical sensor, such as a pH electrode, that produces a signal proportional to analyte concentration. The analyte is normally detected by the biocomponent through a chemical reaction or physical binding. For example, in the case of an enzyme biosensor, a product of the enzyme-catalyzed reaction, such as oxygen, ammonia, hydrochloric acid or carbon dioxide, may be detected by an optical or electrochemical transducer.
Enzymes are preferred biocomponents because they are catalytic, specific to a particular substrate (analyte) and fast acting. Generally, enzymes for use in a biosensor may be disposed within whole cells or extracted from cells and purified. Whole cells are less expensive than purified enzymes and may provide an environment for longer enzyme stability, but cell-based biosensors typically have longer response times and less specificity to a single analyte than purified enzymes due to the presence of multiple enzymes within the cells. Whole-cell biosensors may utilize dead cells or living cells; the later may require proper control of environment and maintenance to retain their efficacy.
The use of purified enzymes in biosensors has also been explored. In D. W. Campbell, “The Development of Biosensors for the Detection of Halogenated Groundwater Contaminants.” Spring 1998, Colorado State University, Fort Collins, Colo., reference is made to a pH optode featuring the reaction illustrated schematically in FIG. 2.4 of Campbell: the cleavage of halide ion X− and proton H+ from a halogenated hydrocarbon by the appropriate hydrolytic dehalogenase. An earlier reference, C. Müller, F. Schubert and T. Scheper, Multicomponent fiberoptical biosensor for use in hemodialysis monitoring, SPIE Proc., Vol. 2131, Biomedical Fiber Optic Instrumentation, Los Angeles, Calif., USA (1994) ISBN 0-8194-1424-7, pp. 555-562, employed a pH optode-type biosensor limited to the use of urease as a catalyst (urea is split into ammonia & CO2): the bifunctional reagent glutaraldehyde was used to bind urease directly to the head of a pH optode. These examples demonstrate the feasibility of utilizing purified enzymes in biosensors with the advantage that the enzymes are not exposed to proteases, found in whole cells, that degrade intracellular proteins. However, extraction, isolation and purification of a particular enzyme can be expensive, tedious and complicated, as well as cause the enzyme to lose a high percentage of its activity.
In addition to the particular circumstances affecting whole cells and purified enzymes discussed above, there are two important challenges to the overall development of enzyme-based biosensors. First, the resolution of similar analytes within a mixture has proven difficult. Although enzymes are generally considered specific, most have activity toward similar molecules within the same chemical family. Second, biosensors containing enzymes that require a cofactor, such as nicotinamide adenine dinucleotide (NADH), have limited lifetimes because cofactors, which are consumed during enzyme-catalyzed detection of an analyte, must be regenerated. The supply of cofactors, either through an ancillary reaction that occurs outside the cell or a metabolic process within a living cell, is non-trivial and has hindered the development of biosensors that require cofactors.